Following the completion of the human genome project, the cataloging of all gene sequences and the acquisition of high-resolution structures of proteins and RNAs, future biological investigations will focus on how the fundamental cellular building blocks interact with each other. Another important issue will be to determine their precise locations in space and time in an attempt to decode and lay out the cell machinery and circuitry. Indeed, many vital functions of the cell are performed by highly organized structures, modular cellular machines that are self-assembled from a large number of interacting macromolecules, and translocated from one cell compartment to another. To unravel the organization and dynamics of these molecular machines in the cell, a tool is needed that can provide dynamic, in vivo, 3D microscopic pictures with nanometer resolution of individual molecules interacting with each other.
Fluorescence microscopy can provide exquisite sensitivity down to the single molecule level for in vitro experiments (1-3). Moreover, it has recently been shown that single fluorophores can be detected in the membrane of living cells with good signal-to-noise ratio (S/N) (4-6). What is not clear yet is whether single molecule fluorescence microscopy can provide the desired spatial and temporal resolution. Technical challenges still to be met are (i) the synthesis of spectrally resolvable, bright and stable fluorophores that can be coupled in vivo to macromolecules; (ii) the development of an easy-to-use and affordable instrument which permits high-resolution localization of individual point-like sources in 3D; and (iii) the ability to perform such measurements at a rate which is compatible with that of biological events. The invention contemplated herein provides an effective solution to the above stated problems.
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Abbreviations used herein include: APD, avalanche photodiode; CL, confidence limit; CLPS, closed-loop piezo-scanner; cpp, count-per-pixel; ICCD, intensified charge-coupled device; NC, nanocrystal; PD, probability distribution; PSF, point-spread function; S/N, signal-to-noise ratio; UHRC, ultra-high-resolution colocalization; TFS, TransFluoSphere, 2D, two-dimensional; 3D, three-dimensional; NSOM, near-field scanning optical microscopy; FWHM, full-width half-maxium.